Abstract

Mutations in CDKN2A account for approximately 40% of familial melanoma cases, and rare mutations in CDK4, BRCA2, BAP1 and in the promoter of TERT also contribute to the disease. However, about half of familial melanoma cases remain unaccounted for. Here we set out to identify high-penetrance susceptibility genes in these unexplained cases.

To achieve this, we sequenced 184 melanoma cases from 105 pedigrees (168 exomes and 16 whole genomes) recruited in the United Kingdom, the Netherlands, and Australia that had been screened and found negative for pathogenetic variants in CDKN2A and CDK4. These patients came from pedigrees with between two and eleven cases of melanoma or were single cases that presented with either multiple primary melanomas, multiple primary cancers, one of which was melanoma, and/or an early age of onset (<4th decade). Analysis of these data showed that these pedigrees carried no mutations in BAP1 or BRCA2.

We found three missense and one splice acceptor mutation, each co-segregating in a different pedigree, in the protection of telomeres 1 (POT1) gene. Importantly, the missense mutations were all located in the highly conserved N-terminal oligonucleotide-/oligosaccharide-binding (OB) domains of POT1, which function to mediate protein – DNA binding. We show that these mutations completely abolish the POT1-DNA complex. Furthermore, we use two methods (one bioinformatic, the other experimental) to assess telomere length in POT1 missense mutation carriers and non-carriers, conclusively showing that individuals with POT1 mutations have substantially longer telomeres than controls. We also amplified and sequenced the POT1 gene product in two of the splice acceptor mutation carriers, showing that the mutation does lead to aberrant splicing.

The families that carry POT1 mutations in this study present not only with melanoma but also with other cancers, namely breast, small cell lung, endometrial and brain tumours, suggesting a possible role for germline POT1 mutations in susceptibility to a range of cancers in addition to melanoma. Furthermore, genotyping across the four identified positions in a melanoma case-control series (1,739 cases and 2,402 controls) revealed that each of two cases carried one of these mutations, whereas no mutations were found in controls, suggesting that POT1 mutations could also account for sporadic melanoma cases.

In this study we describe germline mutations in the gene encoding the telomere-associated protein POT1 in almost 4% of CDKN2A/CDK4-negative familial melanoma pedigrees and in almost 6% of pedigrees with five or more melanoma cases, making POT1 the second most frequently mutated high-penetrance familial melanoma gene reported to date. In combination with the recently described TERT promoter mutation, these findings significantly extend our understanding of a novel mechanism predisposing to the development of familial melanoma. Since the dysregulation of telomere protection by POT1 has recently been identified as a target for potential therapeutic intervention, in the future, it may be possible that early identification of families with POT1 mutations may facilitate better management of their disease.